Embodiments of the present invention relate to a new hyperpolarized imaging agent and to a method of 13C-MR detection, which can be used to determine lactate dehydrogenase (LDH) activity.
Previous studies have demonstrated that hyperpolarized [1-13C]pyruvate metabolism can be imaged in vivo using 13C magnetic resonance spectroscopic imaging (13C MR imaging). For example WO2006/011809 discloses compositions comprising 13C pyruvate and methods for using this in 13C MR imaging.
In tumours, measurements of lactate dehydrogenase (LDH)-catalyzed flux of hyperpolarized 13C label between pyruvate and lactate have been correlated with tumour grade and response to treatment, see e.g. Albers, M. J., et al. Hyperpolarized 13C lactate, pyruvate, and alanine: noninvasive biomarkers for prostate cancer detection and grading. Cancer Res 68, 8607-8615 (2008). Magnetisation transfer measurements in vivo and studies in tumour cell suspensions in vitro have demonstrated unequivocally that isotope exchange between pyruvate and lactate, as opposed to net chemical flux, makes a significant contribution to the observed flux of hyperpolarized 13C label between pyruvate and lactate. This is consistent with the long standing observation that LDH catalyses a reaction that is near-to-equilibrium in the cell and that the mechanism of the enzyme is an ordered ternary complex mechanism, in which the coenzymes NAD+ and NADH bind before pyruvate and lactate respectively. Hence, LDH catalyzes the readily reversible interconversion of pyruvate and lactate with concomitant interconversion of NADH and NAD+, as shown in Scheme 1 below.

Pyruvate is an excellent hyperpolarized substrate for measuring LDH-catalysed flux since it is non-toxic, it polarizes readily to high levels, the polarization is relatively long-lived and transport into the cell is fast. However, pyruvate has a number of important limitations. While it is an endogenous molecule that has shown no evidence of toxicity at the relatively high concentrations used for hyperpolarized 13C imaging experiments in vivo, and in an embodiment lactate is used to measure LDH-catalysed flux, since lactate is present naturally at much higher concentrations than pyruvate and is also transported into the cell very rapidly. A further drawback of using labelled pyruvate to measure LDH activity is that the enzyme is inhibited by the high pyruvate concentrations used for hyperpolarized 13C imaging experiments. However, attempts to use lactate have been relatively unsuccessful since very little label is detected in pyruvate. This is likely because the steady state pyruvate concentration in tissue is very low and therefore there is only a small pool for the hyperpolarized 13C label in lactate to exchange into.
Both Simpson, R. J., Brindle, K. M., Brown, F. F., Campbell, I. D. & Foxall, D. L. A pmr isotope-exchange method for studying the kinetic-properties of dehydrogenases in intact-cells. Biochemical Journal 202, 573-579 (1982), and Simpson, R. J., Brindle, K. M., Brown, F. F., Campbell, I. D. & Foxall, D. L. Studies of lactate-dehydrogenase in the purified state and in intact erythrocytes. Biochemical Journal 202, 581-587 (1982) describe LDH-catalysed exchange of isotope label between pyruvate and lactate. 1H MRS was used in these experiments to measure exchange of methyl deuterated pyruvate ([3-2H3]pyruvate) with protonated lactate in human erythrocyte suspensions. Following addition of methyl deuterated pyruvate and protonated lactate to an erythrocyte suspension, there was an increase in the methyl proton signal from pyruvate and a corresponding decrease in the methyl proton signal from lactate, as deuterium label was exchanged between the two molecules.
Pyruvate inhibition of LDH in these 1H MRS experiments with [3-2H3]pyruvate and protonated lactate was addressed subsequently by instead of measuring label exchange between pyruvate and lactate, exchange of deuterium label was measured between the C2 position of two different isotopically-labelled lactate species, which were distinguished by their methyl label (1H or 2H) (Brindle, K. M. et al. A 1H NMR study of the activity expressed by lactate dehydrogenase in the human erythrocyte. Eur. J. Biochem. 158, 299-305 (1986). This experiment used a spin echo pulse sequence with τ=½J, where J is the 1H-1H coupling constant between the lactate methyl and C2 protons (7.4 Hz) and τ is the delay between the 90 and 180° pulses (68 ms). Under these conditions the lactate methyl resonance is fully inverted in the resulting spectrum. Exchange of the lactate C2 proton for deuterium (D) removes the proton coupling and the phase modulation of the methyl resonance, which now assumes a positive phase. Thus the protonation state of the lactate C2 carbon can be detected via phase modulation of the spin-coupled methyl protons in a homonuclear 1H spin echo experiment. For example, if an equimolar mixture of perdeuterated and protonated lactate is added to a solution containing lactate dehydrogenase and NAD+ then the 1H spectrum will initially show an inverted methyl resonance from the protonated lactate. Following LDH-catalysed exchange of deuterium label at the C2 position between the protonated and deuterated lactate species there will be, at isotopic equilibrium, a mixture of the following lactate species: [3-2H3, 2-21]lactate, [3-2H3, 2-1H1]lactate, [3-1H3, 2-2H1]lactate and [3-1H3, 2-1H1]lactate. Only the methyl protonated species are observed, and of these half will have a proton at the C2 position, and thus the methyl resonance will be inverted, and half will have a deuterium at the C2 position, and thus the methyl resonance will have positive phase. These methyl proton resonances will add to give no signal (there is a slight deuterium shift and thus the signals do not cancel completely). The same experiment can be performed starting with [3-1H3, 2-2H1]lactate and [3-2H3, 2-1H1]lactate. In this case the observed lactate methyl resonance initially has positive phase, due to the C2 deuteron, and this decays to zero following label exchange at the C2 position.
It has now surprisingly been found that experiments using hyperpolarized [1-13C]lactate, in which exchange of 2H label between hyperpolarized [1-13C, 2-2H1]lactate and endogenous unlabelled lactate in a tissue is monitored, can be performed. Embodiments of the present invention propose the use of hyperpolarized [1-13C, 2-2H1]lactate, or other double-labelled isotopomers, to measure LDH activity, where the protonation state of the lactate C2 carbon is detected via phase modulation of the spin-coupled hyperpolarized 13C label at C1, or at other lactate carbons, in a heteronuclear 13C/1H spin echo experiment.
There are disorders indicated by elevated LDH, such as various types of cancer.
Hence, there is a need for new improved methods to determine LDH activity, especially LDH activity in vivo.